Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

B cell–specific loss of histone 3 lysine 9 methylation in the VH locus depends on Pax5

Nature Immunology volume 5, pages 853–861 (2004)Cite this article

Abstract

Immunoglobulin heavy chain rearrangement (VH-to-DJH) occurs only in B cells, suggesting it is inhibited in other lineages. Here we found that in the mouse VH locus, methylation of lysine 9 on histone H3 (H3-K9), a mark of inactive chromatin, was present in non–B lineage cells but was absent in B cells. As others have shown that H3-K9 methylation can inhibit V(D)J recombination on engineered substrates, our data support the idea that H3-K9 methylation inhibits endogenous VH-to-DJH recombination. We also show that Pax5, a transcription factor required for B cell commitment, is necessary and sufficient for the removal of H3-K9 methylation in the VH locus and provide evidence that one function of Pax5 is to remove this inhibitory modification by a mechanism of histone exchange, thus allowing B cell–specific VH-to-DJH recombination.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: H3-K9 methylation associated with the immunoglobulin heavy chain gene segments in Rag1−/− bone marrow pro–B cells, thymocytes and bone marrow–derived macrophages.
Figure 2: DNA methylation is not linked to lineage-specific H3-K9 methylation of VH gene segments.
Figure 3: H3-K9 methylation is inversely correlated to Pax5 expression.
Figure 4: Enforced expression of Pax5 in non–B cell lines results in the removal of H3-K9 methylation at the VH locus.
Figure 5: Accumulation of the histone variant H3.3 at the VH locus correlates with the loss of H3-K9 methylation after Pax5 expression in a double-negative T cell line.

Similar content being viewed by others

References

  1. Rothenberg, E.V. & Anderson, M.K. Elements of transcription factor network design for T-lineage specification. Dev. Biol. 246, 29–44 (2002).

    CAS  PubMed  Google Scholar 

  2. Bassing, C.H., Swat, W. & Alt, F.W. The mechanism and regulation of chromosomal V(D)J recombination. Cell 109, S45–55 (2002).

    CAS  PubMed  Google Scholar 

  3. Kwon, J., Imbalzano, A.N., Matthews, A. & Oettinger, M.A. Accessibility of nucleosomal DNA to V(D)J cleavage is modulated by RSS positioning and HMG1. Mol. Cell 2, 829–839 (1998).

    CAS  PubMed  Google Scholar 

  4. Golding, A., Chandler, S., Ballestar, E., Wolffe, A.P. & Schlissel, M.S. Nucleosome structure completely inhibits in vitro cleavage by the V(D)J recombinase. EMBO J. 18, 3712–3723 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. McBlane, F. & Boyes, J. Stimulation of V(D)J recombination by histone acetylation. Curr. Biol. 10, 483–486 (2000).

    CAS  PubMed  Google Scholar 

  6. Krangel, M.S. Gene segment selection in V(D)J recombination: accessibility and beyond. Nat. Immunol. 4, 624–630 (2003).

    CAS  PubMed  Google Scholar 

  7. Sikes, M.L., Meade, A., Tripathi, R., Krangel, M.S. & Oltz, E.M. Regulation of V(D)J recombination: a dominant role for promoter positioning in gene segment accessibility. Proc. Natl. Acad. Sci. USA 99, 12309–12314 (2002).

    CAS  PubMed  Google Scholar 

  8. Sims, R.J., 3rd, Nishioka, K. & Reinberg, D. Histone lysine methylation: a signature for chromatin function. Trends Genet. 19, 629–639 (2003).

    CAS  Google Scholar 

  9. Osipovich, O., Milley, R., Meade, A., Tachibana, M., Shinkai, Y., Krangel, M.S. & Oltz, E.M. Targeted inhibition of V(D)J recombination by a histone methyltransferase. Nat. Immunol. 5, 309–316 (2004).

    CAS  PubMed  Google Scholar 

  10. Borghesi, L. et al. B Lineage-specific regulation of V(D)J recombinase activity Is established in common lymphoid progenitors. J. Exp. Med. 199, 491–502 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Busslinger, M. Transcriptional control of early B cell development1. Annu. Rev. Immunol. 22, 55–79 (2004).

    CAS  PubMed  Google Scholar 

  12. Nutt, S.L., Urbanek, P., Rolink, A. & Busslinger, M. Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus. Genes Dev. 11, 476–491 (1997).

    CAS  PubMed  Google Scholar 

  13. Urbanek, P., Wang, Z.Q., Fetka, I., Wagner, E.F. & Busslinger, M. Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79, 901–912 (1994).

    CAS  PubMed  Google Scholar 

  14. Hesslein, D.G., Pflugh, D.L., Chowdhury, D., Bothwell, A.L., Sen, R. & Schatz, D.G. Pax5 is required for recombination of transcribed, acetylated, 5′ IgH V gene segments. Genes Dev. 17, 37–42 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Fuxa, M. et al. Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev. 18, 411–422 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Hsu, L.Y., Liang, H.E., Johnson, K., Kang, C. & Schlissel, M.S. Pax5 activates immunoglobulin heavy chain V to DJ rearrangement in transgenic thymocytes. J. Exp. Med. 199, 825–830 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Morshead, K.B., Ciccone, D.N., Taverna, S.D., Allis, C.D. & Oettinger, M.A. Antigen receptor loci poised for V(D)J rearrangement are broadly associated with BRG1 and flanked by peaks of histone H3 dimethylated at lysine 4. Proc. Natl. Acad. Sci. USA 100, 11577–11582 (2003).

    CAS  PubMed  Google Scholar 

  18. Hesslein, D.G. & Schatz, D.G. Factors and forces controlling V(D)J recombination. Adv. Immunol. 78, 169–232 (2001).

    CAS  PubMed  Google Scholar 

  19. Chowdhury, D. & Sen, R. Transient IL-7/IL-7R signaling provides a mechanism for feedback inhibition of immunoglobulin heavy chain gene rearrangements. Immunity 18, 229–241 (2003).

    CAS  PubMed  Google Scholar 

  20. Stanhope-Baker, P., Hudson, K.M., Shaffer, A.L., Constantinescu, A. & Schlissel, M.S. Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro. Cell 85, 887–897 (1996).

    CAS  PubMed  Google Scholar 

  21. Johnson, K., Angelin-Duclos, C., Park, S. & Calame, K.L. Changes in histone acetylation are associated with differences in accessibility of V(H) gene segments to V-DJ recombination during B-cell ontogeny and development. Mol. Cell. Biol. 23, 2438–2450 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Heard, E., Rougeulle, C., Arnaud, D., Allis, C.D. & Spector, D.L. Methylation of histone H3 at Lys-9 is an early mark on the X chromosome during X inactivation. Cell 107, 727–738 (2001).

    CAS  PubMed  Google Scholar 

  23. Love, V.A., Lugo, G., Merz, D. & Feeney, A.J. Individual V(H) promoters vary in strength, but the frequency of rearrangement of those V(H) genes does not correlate with promoter strength nor enhancer-independence. Mol. Immunol. 37, 29–39 (2000).

    CAS  PubMed  Google Scholar 

  24. Angelin-Duclos, C. & Calame, K. Evidence that immunoglobulin VH-DJ recombination does not require germ line transcription of the recombining variable gene segment. Mol. Cell. Biol. 18, 6253–6264 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Souabni, A., Cobaleda, C., Schebesta, M. & Busslinger, M. Pax5 promotes B lymphopoiesis and blocks T cell development by repressing notch1. Immunity 17, 781–793 (2002).

    CAS  PubMed  Google Scholar 

  26. Nutt, S.L., Heavey, B., Rolink, A.G. & Busslinger, M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401, 556–562 (1999).

    CAS  PubMed  Google Scholar 

  27. Rolink, A.G., Nutt, S.L., Melchers, F. & Busslinger, M. Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 401, 603–606 (1999).

    CAS  PubMed  Google Scholar 

  28. Haines, B.B. & Brodeur, P.H. Accessibility changes across the mouse Igh-V locus during B cell development. Eur. J. Immunol. 28, 4228–4235 (1998).

    CAS  PubMed  Google Scholar 

  29. Nussenzweig, M.C. et al. Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin μ. Science 236, 816–819 (1987).

    CAS  PubMed  Google Scholar 

  30. Yu, D., Allman, D., Goldschmidt, M.H., Atchison, M.H., Monroe, J.G. & Thomas-Tikhonenko, A. Oscillation between B-lymphoid and myeloid lineages in Myc-induced hematopoietic tumors following spontaneous silencing/reactivation of the EBF/Pax5 pathway. Blood 101, 1950–1955 (2003).

    CAS  PubMed  Google Scholar 

  31. Yu, D. & Thomas-Tikhonenko, A. A non-transgenic mouse model for B-cell lymphoma: in vivo infection of p53-null bone marrow progenitors by a Myc retrovirus is sufficient for tumorigenesis. Oncogene 21, 1922–1927 (2002).

    CAS  PubMed  Google Scholar 

  32. Heavey, B., Charalambous, C., Cobaleda, C. & Busslinger, M. Myeloid lineage switch of Pax5 mutant but not wild-type B cell progenitors by C/EBPα and GATA factors. EMBO J. 22, 3887–3897 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Rolink, A.G., Winkler, T., Melchers, F. & Andersson, J. Precursor B cell receptor-dependent B cell proliferation and differentiation does not require the bone marrow or fetal liver environment. J. Exp. Med. 191, 23–32 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Janicki, S.M. et al. From silencing to gene expression: real-time analysis in single cells. Cell 116, 683–698 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Ahmad, K. & Henikoff, S. The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell 9, 1191–1200 (2002).

    CAS  PubMed  Google Scholar 

  36. Ahmad, K. & Henikoff, S. Histone H3 variants specify modes of chromatin assembly. Proc. Natl. Acad. Sci. USA 99, 16477–16484 (2002).

    CAS  PubMed  Google Scholar 

  37. Tagami, H., Ray-Gallet, D., Almouzni, G. & Nakatani, Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116, 51–61 (2004).

    CAS  PubMed  Google Scholar 

  38. Schlissel, M., Corcoran, L. & Baltimore, D. Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription. J. Exp. Med. 173, 711–720 (1991).

    CAS  PubMed  Google Scholar 

  39. Chowdhury, D. & Sen, R. Stepwise activation of the immunoglobulin mu heavy chain gene locus. EMBO J. 20, 6394–6403 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Su, I.H. et al. Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement. Nat. Immunol. 4, 142–131 (2002).

    CAS  Google Scholar 

  41. Kosak, S.T. et al. Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296, 158–162 (2002).

    CAS  PubMed  Google Scholar 

  42. Corcoran, A.E., Riddell, A., Krooshoop, D. & Venkitaraman, A.R. Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 391, 904–907 (1998).

    CAS  PubMed  Google Scholar 

  43. Costa, T.E., Suh, H. & Nussenzweig, M.C. Chromosomal position of rearranging gene segments influences allelic exclusion in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 2205–2208 (1992).

    CAS  PubMed  Google Scholar 

  44. ten Boekel, E., Melchers, F. & Rolink, A.G. Changes in the V(H) gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 7, 357–368 (1997).

    CAS  PubMed  Google Scholar 

  45. Ji, Y., Zhang, J., Lee, A.I., Cedar, H. & Bergman, Y. A multistep mechanism for the activation of rearrangement in the immune system. Proc. Natl. Acad. Sci. USA 100, 7557–7562 (2003).

    CAS  PubMed  Google Scholar 

  46. Mostoslavsky, R. et al. κ chain monoallelic demethylation and the establishment of allelic exclusion. Genes Dev. 12, 1801–1811 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Whitehurst, C.E., Schlissel, M.S. & Chen, J. Deletion of germline promoter PDβ1 from the TCRβ locus causes hypermethylation that impairs Dβ1 recombination by multiple mechanisms. Immunity 13, 703–714 (2000).

    CAS  PubMed  Google Scholar 

  48. Durum, S.K. et al. Interleukin 7 receptor control of T cell receptor γ gene rearrangement: role of receptor-associated chains and locus accessibility. J. Exp. Med. 188, 2233–2341 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Cherry, S.R. & Baltimore, D. Chromatin remodeling directly activates V(D)J recombination. Proc. Natl. Acad. Sci. USA 96, 10788–10793 (1999).

    CAS  PubMed  Google Scholar 

  50. Bannister, A.J., Schneider, R. & Kouzarides, T. Histone methylation: dynamic or static? Cell 109, 801–806 (2002).

    CAS  PubMed  Google Scholar 

  51. Bolland, D.J. et al. Antisense intergenic transcription in V(D)J recombination. Nat. Immunol. 5, 630–637 (2004).

    CAS  PubMed  Google Scholar 

  52. Orphanides, G. & Reinberg, D. RNA polymerase II elongation through chromatin. Nature 407, 471–475 (2000).

    CAS  PubMed  Google Scholar 

  53. Chang, D., Angelin-Duclos, C. & Calame, K. BLIMP-1: trigger for differentiation of myeloid lineage. Nat. Immunol. 1, 169–176 (2000).

    CAS  PubMed  Google Scholar 

  54. Kouro, T., Medina, K.L., Oritani, K. & Kincade, P.W. Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators. Blood 97, 2708–2715 (2001).

    CAS  PubMed  Google Scholar 

  55. Grunau, C., Clark, S.J. & Rosenthal, A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids. Res. 29, E65–65 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. Busslinger (Reasearch Institute of Molecular Pathology, Vienna, Austria) for the gift of the Pax5−/− mice; P. Kincade (Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma) for direction in establishing the culture conditions used for the Pax5−/− cells; C. Wickens for assistance in maintaining the mouse colonies; C. Tunyaplin and Y. Zou for critical reading of the manuscript; and D. Spector for advice regarding controls for the H3.3 analyses. Supported by National Institutes of Health (R01 AI43576, R01 AI32524, R01 CA102709 and GM40924).

Author information

Authors and Affiliations

  1. Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, 10032, New York, USA

    Kristen Johnson, Kuo-I Lin & Kathryn Calame

  2. Section of Immunobiology, Yale University of Medicine, Box 208011, 310 Cedar Street, New Haven, 06520-8011, Connecticut, USA

    David L Pflugh, Alfred L M Bothwell & David G Schatz

  3. Department of Pathobiology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, 19104-6051, Pennsylvania, USA

    Duonan Yu & Andrei Thomas-Tikhonenko

  4. Department of Cell Biology, Yale University of Medicine, Box 208011, 310 Cedar Street, New Haven, 06520-8011, Connecticut, USA

    David G T Hesslein

  5. Howard Hughes Medical Institute, Yale University of Medicine, Box 208011, 310 Cedar Street, New Haven, 06520-8011, Connecticut, USA

    David G Schatz

  6. Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, 10032, New York, USA

    Kathryn Calame

Authors
  1. Kristen Johnson
    View author publications

    Search author on:PubMed Google Scholar

  2. David L Pflugh
    View author publications

    Search author on:PubMed Google Scholar

  3. Duonan Yu
    View author publications

    Search author on:PubMed Google Scholar

  4. David G T Hesslein
    View author publications

    Search author on:PubMed Google Scholar

  5. Kuo-I Lin
    View author publications

    Search author on:PubMed Google Scholar

  6. Alfred L M Bothwell
    View author publications

    Search author on:PubMed Google Scholar

  7. Andrei Thomas-Tikhonenko
    View author publications

    Search author on:PubMed Google Scholar

  8. David G Schatz
    View author publications

    Search author on:PubMed Google Scholar

  9. Kathryn Calame
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Kathryn Calame.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1 (download PDF )

H3K4 methylation of immunoglobulin heavy chain gene segments in pro-B and DN T cells. (PDF 643 kb)

Supplementary Fig. 2 (download PDF )

H3K4 Methylation does not change during the process of allelic exclusion. (PDF 249 kb)

Supplementary Table 1 (download PDF )

Primers. (PDF 9 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Johnson, K., Pflugh, D., Yu, D. et al. B cell–specific loss of histone 3 lysine 9 methylation in the VH locus depends on Pax5. Nat Immunol 5, 853–861 (2004). https://doi.org/10.1038/ni1099

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/ni1099

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing